Targeted Osmotic Lysis of Cancer Cells

ABSTRACT

A targeted osmotic lysis (TOL) of tumor cells that over-express voltage-gated sodium channels (VGSCs) has been developed that uses a combined therapy of a drug that blocks sodium, potassium-adenosine triphosphatase (Na + , K + -ATPase) that is then followed by an activation of VGSCs, for example, by electrical or pharmacological stimulation. Activation of VGSCs conducts sodium into the cancer cells in much greater amounts than non-cancer cells. Water follows this sodium gradient into the cancer cells, causing swelling and lysis. Because non-cancerous cells do not over-express VGSCs, less sodium and less water will enter the cells, and the non-cancerous cells will not lyse. This method is applicable to all cells that over-express VGSCs, including, but not limited to, highly invasive breast cancer, prostate cancer, small cell lung cancer, non-small cell lung carcinoma, lymphoma, mesothelioma, neuroblastoma, and cervical cancer.

This is a continuation of application Ser. No. 13/552,909, filed Jul.19, 2012, now allowed with the issue fee paid; which claimed the benefitof the filing date of provisional U.S. application Ser. No. 61/510,258,filed Jul. 21, 2011 under 35 U.S.C. §119(e).

TECHNICAL FIELD

This invention pertains to a method to target cancer cells thatover-express voltage-gated sodium channels (VGSCs or “sodium channels”)and to cause osmotic lysis of these cancer cells by initially inhibitingthe sodium, potassium-adenosine triphosphatase (Na⁺, K⁺-ATPase or“sodium pump”), and then stimulating the VGSCs to cause sodium and waterto enter the cancer cells.

BACKGROUND ART

Chemotherapy and radiotherapy of metastatic cancer, because of toxicityto both normal and abnormal tissues, present the clinician with thedifficult challenge of trying to kill the neoplastic disease beforekilling the patient; a balance between treatment and rescue. Alltraditional cancer treatments are associated with toxicity, an increasein morbidity, and a reduction in quality of life that may extend farbeyond the period of treatment. A major focus of current anti-neoplastictreatments is targeting treatment to the cancer cells, for example,targeting proteins expressed or over-expressed by cancer cells, but notby normal tissue.

Many invasive cancer cell types over-express voltage-gated sodiumchannels (VGSCs; or “sodium channels”) by more than 1000-fold greaterthan normal cells (1, 2, 7). Cancer cells that over-express VGSCs areepithelial carcinomas that include, but are not limited to, highlyinvasive breast cancer (4, 10, 13, 27), prostate cancer (2, 6, 7, 8, 18,19, 20, 21, 22, 26), small cell lung cancer (3, 23), non-small cell lungcarcinoma (28), lymphoma (9), neuroblastoma (25), and cervical cancer(5). Mesothelioma which is not classified as an epithelial cancer isalso known to over-express VGSCs (12). When these sodium channels areactivated, Na⁺ is conducted into the cells. In these cancers, the degreeof metastasis is directly related to an increased expression of VGSCs(1, 7; see also U.S. Pat. No. 7,393,657). Physiologically, these cancercells share certain cellular properties with normal excitable cells suchas neurons and cardiac myocytes (for example, the conduction of actionpotentials). U.S. Pat. No. 7,393,657 discloses the use of inhibitors ofVGSCs as a treatment for cancer, including breast cancer.

Of the 1.6 million people contracting epithelial cell cancer each yearin the U.S., 40% are considered to be “highly invasive” and over-expressVGSCs (10). These patients diagnosed with malignant/metastaticcarcinomas are treated currently with major and often disfiguringsurgical procedures, chemotherapy and/or radiation. More than 400,000people die from epithelial cell carcinoma each year in the United Statesand an estimated 10 times that world-wide. In addition, another1,200,000 U.S. patients diagnosed with invasive cancer are successfullytreated with traditional surgery, chemotherapy and/or radiation. Breastcell carcinoma is an example of a highly invasive cancer. More than40,000 people die from breast cell carcinoma each year in the UnitedStates and 465,000 world-wide. Greater than 90% of these deaths are dueto metastasis of the primary tumor. In addition, another 170,000 U.S.women diagnosed with invasive breast cancer are successfully treatedwith traditional mastectomy, lumpectomy, chemotherapy and/or radiation.Of the 207,000 people contracting breast cancer each year, 40% of thecancers are considered to be “highly invasive”, and over-express VGSCs(10).

The family of sodium channels named “voltage-gated sodium channels” wasso designated due to the sensitivity to small changes (>40 mV) in thevoltage gradient across the cellular membrane. They have also been shownto be activated by many forms of stimulation—mechanical disturbances inthe membrane, ultrasound (29), magnetic fields (29), and several drugs.There are nine members of the VGSC family, with variants of many of theisoforms. They are designated Na_(v)1.X, where X represents 1-9.Subtypes are designated with a letter a, b, etc.

Na⁺, K⁺-ATPase is a ubiquitous transmembrane protein in animal cells,and functions to maintain an ion imbalance across the cell membranewhere more charged ions are located outside of the cell, largely sodiumions, than inside. This produces an electrochemical gradient that is inhomeostatic balance. When ionic imbalance shifts in the presence of achange in voltage an action potential is generated causing a transientosmotic shift toward an intracellular hypertonic state. The restorationof the sodium imbalance is an essential function performed by Na⁺,K⁺-ATPase. When Na⁺, K⁺-ATPase does not function properly, water followssodium into the cell to restore osmotic balance thereby increasing cellvolume. In normal cells this shift in cell volume is tolerated due tomembrane compliance. Blocking Na⁺, K⁺-ATPase function can lead to a lossof cellular excitability and an increase in cellular volume. Manyinhibitors are known, including the cardiac glycosides. The isozymesvary in their sensitivity to each of the cardiac glycoside drugs. Morethan 30 drugs have been shown to inhibit sodium pump activity. Theseinclude ouabain, digitalis and its active ingredients digoxin anddigitoxin.

U.S. Patent Application Publication No. 2007/0105790 discloses the useof cardiac glycosides (e.g., ouabain and proscillaridin) either alone orin combination with other standard cancer therapeutic agents to treatpancreatic cancers by causing cell apoptosis.

U.S. Patent Application Publication No. 2009/0018088 discloses the useof cardiac glycosides, including digoxin and ouabain, to induce cellapoptosis as a treatment for cancer.

DISCLOSURE OF INVENTION

We have discovered that, in cancer cells that express excess VGSCs, ifthe Na⁺, K⁺-ATPase (sodium pump) is blocked, and then VGSCs areactivated, the cells will lyse and die. The activation of the VGSCscauses Na⁺ to be conducted into the cells, but due to the inhibition ofthe sodium pumps, the Na⁺ cannot be pumped back out of the cell. Becausewater flows into the cell based on a Na⁺ gradient, water flowing intothe cell causes the cells that over-express Na⁺ channels (i.e., allowmore Na⁺ into the cells) to swell and burst when membrane compliance isexceeded. Because a lesser amount of Na⁺ enters normal cells that do notover-express VGSCs, normal tissue does not swell or lyse. We have calledthis two-stage treatment “Targeted Osmotic Lysis” (TOL), and have shownthat this treatment is effective in treating highly invasive cancercells. In addition, we have shown that some highly invasive cancer cellsover-express Na⁺, K⁺-ATPase (the sodium pump) to compensate for theincrease in Na⁺ influx through the over-expressed VGSCs (unpublished;Data not shown). For example, MCF-7 breast cancer cells over-expressNa⁺, K⁺-ATPase by 8- to 10-fold, whereas MDA-MB-231 breast cancer cellsover-express Na⁺, K⁺-ATPase by only 2-fold.

In summary, we have demonstrated efficacy of TOL in both in vitro and invivo models of invasive carcinoma. We have shown the usefulness of bothelectrical and pharmacological stimulation of the cancer cells to induceTOL. We have demonstrated TOL is effective in seven cell lines derivedfrom four different tissue types. As little as 100-fold increase insodium channel expression compared to normal tissue is sufficient toconfer susceptibility to TOL treatment, although time-to-lysis isinversely related to extent of sodium channel expression. We havedemonstrated in vivo that TOL does not affect normal tissue, even inthose tissues that normally express relatively high concentrations ofsodium channels. Finally, we have demonstrated that TOL can be inducedusing any drug or process that blocks sodium pumps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effects on cell viability of cultured breastcancer cells from a control and treatment with ouabain (a sodium pumpblocker) by itself, treatment with veratridine (sodium channelstimulator) by itself, and treatment with a combination of ouabain andveratridine (Targeted Osmotic Lysis).

FIG. 2 illustrates the change in tumor volume over time of breast cancerxenografts in mice following a single treatment on Day 0 with saline andno electrical stimulation (Saline-No Stim), with saline and electricalstimulation (Saline-Stim), with ouabain and no stimulation (Ouabain-NoStim), or with ouabain and electrical stimulation (Ouabain-Stim).

MODES FOR CARRYING OUT THE INVENTION

We have developed a targeted osmotic lysis (TOL) of tumor cells thatover-express VGSCs by a combined therapy of a drug that blocks Na⁺,K⁺-ATPase that is then followed by an activation of VGSCs, for example,by electrical, magnetic, ultrasound (29), or pharmacologicalstimulation. Activation of VGSCs conducts sodium into the cancer cellsin much greater amounts than non-cancer cells. Water follows this sodiumgradient into the cancer cells, causing swelling and lysis. Becausenon-cancerous cells do not over-express VGSCs, less sodium and lesswater will enter the normal cells, and the cells will not lyse.

This method is applicable to all cancer cells that over-express VGSCs.The cancers that over-express VGSCs can be identified through theliterature or by assaying the cancer cells for VGSCs by methods known inthe art (for example, U.S. Patent Application Publication 2009/0074665).For example, all epithelial cancer cells assayed to date have been shownto over-express VGSCs, including but not limited to, highly invasivebreast cancer (4, 10, 13, 27), prostate cancer (2, 6, 7, 8, 18, 19, 20,21, 22, 26), small cell lung cancer (3, 23), non-small cell lungcarcinoma (28), lymphoma (9), neuroblastoma (25), and cervical cancer(5). Mesothelioma which is not classified as an epithelial cancer isalso known to over-express VGSCs (12). Since all epithelial cancersstudied to date have been shown to over-express VGSCs, other cancersthat are classified as epithelial cell carcinomas are expected toover-express VGSCs, including but not limited, to gliomas, neuromas,hepatic cancer, ovarian cancer, bladder cancer, pancreatic cancer,thyroid cancer, splenic cancer, stomach cancer, cervical cancer, skincancers, testicular cancer, renal cancer, and oral cancers.

The Na⁺, K⁺-ATPase blocker may be delivered to a single tumor via director intravenous administration, to a single organ or area via intravenousor intraluminal administration, or the entire body via intravenous,subcutaneous intramuscular or oral administration. Electrical orpharmacological stimulation of sodium channels can be delivered to asingle tumor, a single organ, a section of the body, or the entire body.Theoretically, all types and subtypes of the VGSCs family should beequally susceptible to this technology. To date, the cell lines testedover-express Na_(v)1.5, Na_(v)1.5a and Na_(v)1.7, all of which mediatedtargeted lysis.

Examples of pharmaceutical compounds that can be used to block Na⁺,K⁺-ATPase are numerous. See, for example, U.S. Published PatentApplications No. 2007/0105790 and 2009/0018088. These compounds include,but are not limited to, the following: ouabain (g-Strophantin);dihydroouabain; ouabain octahydrate; ouabagenin; digoxin; digitoxin;digitalis; acetyldigitoxin; acetyldigoxin; lanatoside C; deslanoside;metildigoxin; gitoformate; oleandrin; oleandrigenin; bufotoxin;bufotalin; marinobufagenin (3,5-dihydroxy-14,15-epoxy bufodienolide);palytoxin; oligomycins A, B, C, E, F, and G; rutamycin (oligomycin D);rutamycin B; strophanthin (g-strophanthin, Acocantherine);k-β-strophanthin; strophanthidin; k-strophanthoside; cymarin;erysimoside (cardenolide); helveticoside; peruvoside; hypothalamic Na.,K.-ATPase inhibitory factor (HIF); the aglycone of HIF; arenobufagin;cinobufagin; marinobufagin; proscillaridin; scilliroside;daigremontianin; and all other inhibitors of Na⁺, K⁺-ATPase,combinations and derivatives of each.

Methods to electrically stimulate tumor tissue are well known in the art(See, for example, U.S. Pat. No. 7,742,811). Some examples include, butare not limited to, the following: use of direct or alternating current(DC or AC); use of a direct application of electrodes to a tumor; use ofa direct application of electrodes to an organ with multiple metastases;a transcutaneous electrical stimulation using deep muscle stimulator; atranscutaneous electrical nerve stimulation (“TENS”) unit or similar;and whole-body electrical stimulation with a voltage no less than 40 mVand preferably about 1 V. In addition, magnetic fields and ultrasoundhave been used to stimulate nervous tissue, and sodium pumps (29).

Examples of pharmaceutical compounds that are known to increase theactivity of VGSCs are known in the art. Examples include, but are notlimited to, the following: veratridine; veracevine; antillatoxin (ATX);ATX II; batrachotoxin; aconitine; grayanotox; Grayanotoxin IIIHemi(ethyl acetate); Antillatoxinn; Nigericin; gramicidin;α-Pompilidotoxin; 0-Pompilidotoxin; Hoiamide A; brevetoxin (PbTx-2);ciguatoxins; scorpion neurotoxin; BDF 9148; DPI 201-106; TC0101029(SCNM1); Cypermethrin; Alphamethrin; palytoxin; and all combinations andobvious derivatives of each of the above.

We expect that the Targeted Osmotic Lysis technique will: (1) increasethe survival rate of patients with highly invasive tumors; (2) reducethe number of radical mastectomies and lumpectomies for breast cancerpatients; (3) reduce the extent of morbidity due to treatment; (4)reduce recovery time from treatment; and (5) be applicable to allcarcinomas that over-express VGSCs.

We have shown in in vivo TOL experiments described below a 30-40%survival rate in a breast cancer mouse model, and the in vitroexperiments suggest a higher success rate once parameters are optimized.The limiting factor of in vivo TOL is the delivery efficiency of theinhibitor of the sodium pump to the tumor cells. In the initial pilotstudy, the tumors in which the tumors were highly vascularized and thusdelivery of the inhibitor was more efficient were lysed following TOLtreatment. In contrast, tumors with little vascularization remainedviable. With further drug delivery improvement, it is believed thatnearly all cancers that over-express VGSCs (e.g., 40% of all breastcancers) will be treatable with TOL, and in the case of breast cancers,mastectomies and lumpectomies should be reduced by up to 40%. This willalso reduce the need for reconstructive surgery.

Targeted Osmotic Lysis is expected to have many advantages overtraditional cancer therapies. Chemotherapy typically causes damage inhealthy, as well as cancerous, tissue, leading to lengthy recovery andchronic morbidity. By comparison, TOL will destroy only cells thatover-express VGSCs. Thus, a more selective lesion of diseased tissue isexpected. This will contribute to fewer long-term adverse effects oftreatment. Radiation therapy is typically directed to kill the healthytissue surrounding the cancerous tissue. Like chemotherapy, this oftenleads to lengthy recovery and chronic morbidity. Because of theselectivity of TOL for cells that over-express VGSCs, there is little tono peri-neoplastic damage.

The adverse effects of chemotherapy and radiation therapy (“RT”) arewell documented, and treatment compliance is often problematic. Webelieve that TOL of carcinoma, when optimized, will require only one ortwo treatments, each lasting a few hours, with about a 2-5 week recoveryfrom each. Because the adverse effects of the treatment are expected tobe minimal compared with traditional therapies, treatment compliance isexpected to be improved over traditional therapies.

Another current problem with chemotherapy and radiotherapy (RT) is thelong term or permanent damage that results from these treatments.Chemotherapy is known to produce necrotic and demyelinatingneuropathies, memory changes, sexual and fertility changes. Long-termadverse effects of RT vary widely with treatments, but are known toproduce various neuropathies and chronic pain, motor deficits, andcognitive deficits, for whole-brain radiation. With TOL, fewer chronicadverse effects are expected and quality of life will be improved formost patients as compared to traditional treatments.

Recovery from chemotherapy and/or radiation therapy typically takesmonths. The recovery from a TOL treatment will involve the resorption ofthe dead tissue, and will manifest as fever and other flu-like symptoms.The degree of the fever and other symptoms will vary with the degree ofmetastasis and size of the tumors. The fever and joint pain can bealleviated with common analgesic-antipyretic treatments (acetaminophen,NSAIDs, etc.) Therefore, quality of life immediately after treatmentwill likely be greater than with traditional treatments.

Two possible adverse reactions to TOL might occur. In rats, anover-expression of Na_(v)1.7 VGSCs in dorsal root ganglia has beenassociated with inflammation (15, 16, 17). Thus, in patients that havemajor inflammatory diseases, such as rheumatoid arthritis, Crohn'sdisease, or infection, TOL might potentially produce damage to theperipheral nervous system. The second possibility of an adverse reactionmight be the development of an autoimmune reaction to the proteinsreleased as the cells lyse. As cancer cells lyse, there is the potentialfor abnormal proteins to be released that are recognized by T-cells asalien to the patient. This side effect has been noted forchemotherapeutic agents and radiation treatments that cause nonspecificlysis of cells.

One embodiment of this technology is a method to lyse metastatic tumorcells that have increased expression of VGSCs over that seen innon-tumor cells, said method comprising the following two steps: Step 1is to administer to the tumor cells a compound that inhibits theactivity of Na⁺, K⁺-ATPase; and Step 2 is to administer to the tumorcells a compound that increases the activity of the VGSCs. Thecombination of these two steps will cause the tumor cells to take up asurplus of sodium, which in turn will cause water to flow into thecells. The tumor cells will swell and eventually lyse due to the excesswater.

A second embodiment of this technology is a method to lyse metastatictumor cells that have increased expression of VGSCs over the activityseen in non-tumor cells, said method comprising the following steps:Step 1 is to administer to the tumor cells a compound that inhibits theactivity of Na⁺, K⁺-ATPase; and Step 2 is to administer to the tumorcells an electrical stimulation to increase the activity of the VGSCs.Again, similar to above, the tumor cells will take in a surplus ofsodium causing water to flow into the cells, and will swell andeventually lyse.

A third embodiment of this technology is a method to lyse metastatictumor cells that have increased expression of VGSCs over the activityseen in non-tumor cells, said method comprising the following steps:Step 1 is to administer to the tumor cells a compound that inhibits theactivity of Na⁺, K⁺-ATPase; and Step 2 is to administer to the tumorcells a magnetic stimulation to increase the activity of the VGSCs.

A fourth embodiment of this technology is a method to lyse metastatictumor cells that have increased expression of VGSCs over the activityseen in non-tumor cells, said method comprising the following steps:Step 1 is to administer to the tumor cells a compound that inhibits theactivity of Na⁺, K⁺-ATPase; and Step 2 is to administer to the tumorcells an ultrasound stimulation to increase the activity of the VGSCs.

A fifth embodiment is a method to identify metastatic tumor cells thatwould be lysed by the above embodiments, comprising assaying the tumorcells for the degree of expression of VGSCs and comparing the degree ofexpression with that from normal, non-tumor cells; wherein themetastatic tumor cells that would be lysed using the above methods wouldbe tumor cells with a higher degree of expression for VGSCs.

EXAMPLE 1 Targeted Osmotic Lysis in Breast Cancer Cells

The effectiveness of TOL has been tested using an in vitro model forbreast cancer. MDA-MB-231 breast cancer cells were cultured to completeconfluency in 96-well plates. These breast cancer cells are known toover-express VGSCs by 1400-fold. These cells were exposed to Dulbecco'smodified Eagle's medium (DMEM), or the cardiac glycoside drugs (knownsodium pump inhibitors) ouabain (10 pM-100 nM; Sigma Chemical Co., St.Louis, Mo.) or digitoxin (100 pM-1 μM; Sigma) dissolved in DMEM for 30min. After exposure to the sodium pump inhibitors, an electric current(0 V, 100 mV, or 1 V DC) was passed across the cells using an anode anda cathode placed touching the bottom of each well or an etched circuitin the bottom of the plate. The electric current was generated using aGrass Model SD9 stimulator (Grass Instruments, Quincy, Mass.) throughplatinum wire electrodes. In 6 of 24 wells that cells were exposed to ≧1nM ouabain or ≧10 nM digoxin, and exposed to 100 mV or 1 V electriccurrent, every cancer cell died (Data not shown).

EXAMPLE 2 Targeted Osmotic Lysis Using In Vivo Model of Cancer

As an in vivo model of cancer, MDA-MB-231 cells were suspended inMatrigel and injected subcutaneously into the backs of 5 nude (J-NU)mice. Each mouse developed 0.75-1.2 cm tumors in 3-5 weeks. The micewere then injected subcutaneously with 10 mg/kg ouabain or saline. After30 min, the mice were anesthetized with 4% isoflurane, and the tumorsexposed through a small incision in the skin. An anode and a cathodewere inserted into each tumor, and a train of 120 1 V DC pulses (10 ms,2 Hz for 1 min) was delivered through the anode and cathode as discussedabove. A total of 11 tumors were tested. Three tumors were from micetreated with ouabain and electrically stimulated (O-ES experimental). Ofthe rest, two were from a mouse treated with saline and not electricallystimulated (S-NS control), three were from mice treated with ouabain butnot electrically stimulated (O-NS control), and three were from micetreated with saline and electrically stimulated (S-ES control). Theelectrical stimulation was repeated 15 and 30 min later. One day later,mice were sacrificed with an overdose of Nat pentobarbital, perfusedwith 4% buffered paraformaldehyde, and the tumors removed. The tumorswere sectioned at 5 μm and stained with hematoxylin and eosin. Of thethree tumors in the experimental group (the O-ES group), the tumor fromone mouse showed 80% cell death after treatment. However, normal muscletaken from the base of the tumor in the same mouse showed no sign ofcell death. The other two mice in the O-ES group had tumors that werenot highly vascularized, and thus the ouabain could not efficientlydistribute to the tumor cells. Consequently, no lysis was seen in thetumors of these two mice. None of the tumors in any of the other threetreatments showed any sign of cell lysis.

EXAMPLE 3 In Vitro Targeted Osmotic Lysis of Multiple Cell Lines ofCarcinoma

Initially we cultured MDA-MB-231 (ATCC, Manassas, Va., cat #HTB-26)breast cancer cells to complete confluency in 96-well plates. Thesecells over-express VGSCs by 100-fold. These cells were exposed toDulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island,N.Y.), or the cardiac glycoside drugs ouabain (10 pM-100 nM) or digoxin(100 pM-1 μM) dissolved in DMEM for 30 min. An electric current (0 V,100 mV, or 1 V DC) was passed across the cells by means of an anode anda cathode placed touching the bottom of each well as in Example 1. In 6of 24 wells that were exposed to ≧1 nM ouabain or ≧10 nM digoxin, allcancer cells died. Of these six wells, 1 well was exposed to 10 nMouabain and 1 VDC, 1 was treated with 100 nM ouabain with 100 mV DC, 1was treated with 100 nM ouabain and with 1V DC, 2 were treated with 100nM digoxin and with 1V DC, and 1 treated with 1 μM digoxin and 100 mVDC.

We also used two cardiac glycoside drugs that are currently FDA-approvedfor clinical use, ouabain and digoxin, for in vitro lysis of multiplelines of carcinoma. To demonstrate that TOL is effective for anycarcinoma that over-expresses VGSCs and that any sodium pump blockercould be used, we cultured seven cancer cell lines derived from tissuetypes representing four of the most common deadly cancers. Cell lineswere: MDA-MB-231 (breast cancer); MCF-7 (breast cancer; ATCC# HTB-22);LNCaP (prostate cancer; ATCC# CRL-1740); DU145 (prostate cancer; ATCC#HTB-81); MCA-38 (colon cancer; from Dr. Augusto Ochoa); A549 (non-smallcell lung cancer; ATCC# CCL-185); and 3LL (non-small cell lung cancer;from Dr. Augusto Ochoa, Louisiana State University, New Orleans, La.).Cells were plated in 35 mm diameter culture dishes in Dulbecco'sModified Eagle's Medium (DMEM, Gibco) supplemented with 4 mML-glutamine, 1 mM sodium pyruvate and 10 μM insulin for 18-24 hr. Cellswere then incubated for 15-45 min in 100 nM ouabain, 1 μM digoxin or nodrug. A 2 V DC current, 200 pulses per second was passed acrossindividual cells using a Grass Model SD9 stimulator (Grass Instruments,Quincy, Mass.) through platinum wire electrodes held in place with aDavid Kopf Instrument micromanipulator (Tarzana, Calif.), and the timeto lysis was measured. Video recordings of representative experimentswere prepared digitally using a Canon Vixia HFM400 camera attached toLeica DM IL microscope, and are available upon request. Meantime-to-lysis (±SEM) for each cell type and for each drug expressed insec are presented in Table 1. As shown in Table 1, none of the controlslysed within the 5 min time limit. All of the cancer cells lysed, withthe time to lysis faster for the cells with the largest known expressionof VGSCs.

TABLE 1 Time to Lysis of Cultured Cancer Cells. Seven cell lines ofcarcinoma originating from four different cancer types were incubated inmedia alone or media + ouabain (100 nM) or media + digoxin (1 μM), andeither stimulated or not stimulated. Time to lysis for a 5 min timeperiod was determined by appearance of extracellular cytosol extendingfrom the base of the cell or a visible split in the cell surface. Meantime-to-lysis ± SEM was calculated for each group and expressed inseconds. None of the cells from control conditions lysed within the 5min time limit. Therefore, a value of 300 sec was used for all controlsfor statistical purposes. All drug + stimulation values weresignificantly different from controls (p < .01; 2-way ANOVA). VGSC datafor the colon and lung cancer cells were not available. VGSC over-Ouabain + Digoxin + All expres- Stimulation Stimulation Controls sion*(sec) (sec) (sec) Breast Cancer: MDA-MB-231 ++++ 69.3 ± 4.82 68.39 ±7.31 300 (n = 44) (n = 33) (n = 60) MCF-7 ++ 170.5 ± 15.35 180.3 ± 9.07300 (n = 32) (n = 10) (n = 56) Prostate Cancer: LNCaP +++ 143.1 ± 5.78 122.9 ± 3.13 300 (n = 28) (n = 21) (n = 32) DU145 +++ 81.6 ± 3.96 111.3± 9.7  300 (n = 30) (n = 21) (n = 26) Colon Cancer: MCA-38 n/a 74.0 ±1.0  122.7 ± 9.02 300 (n = 2)   (n = 3) (n = 3)  Lung Cancer: A549 n/a149.9 ± 11.70  179.8 ± 10.82 300 (n = 22) (n = 22) (n = 18) 3LL n/a115.9 ± 3.33  163.75 ± 8.27  300 (n = 31) (n = 24) (n = 15) *Values from(24); + = ≧100-fold, + + = ≧250-fold, +++ = ≧500-fold, ++++ =≧1,000-fold over-expression of sodium channels.

EXAMPLE 4 In Vitro TOL of Breast Cancer Using PharmacologicalStimulation of VGSCs

The effect of the combination of ouabain (a sodium pump inhibitor) andveratridine (a sodium pump stimulator) on viability of culturedMDA-MB-231 and MCF-7 breast cancer cells was assessed. The former cellsover-express VGSCs by more than 1000-fold, whereas the latter cellsover-express VGSCs by 100-fold (24). MDA-MB-231 cells were cultured inDMEM, whereas MCF-7 cells were cultured in DMEM+10 μM insulin. Aftercentrifugation, both cell lines were re-suspended in DMEM+10 μM insulinto assure normal functioning of Na⁺, K⁺-ATPase, and approximately 5000cells were added to each well in a 96-well plate in a volume of 100 μl.After 18 hr, the media was aspirated, and the cells were treated with100 μl DMEM+10 μM insulin (“media”) alone, media+100 nM ouabain,media+30 μM veratridine, or media+100 nM ouabain+30 μM veratridine.After 1 hr, 10 μl alamar blue was added to each well, and cell viabilitydetermined 4 hr after this addition. All determinations were obtained inquadruplicate, and the experiment repeated three times. The results areshown in FIG. 1. In MDA-MB-231 cells, ouabain alone produced no decreasein cell viability compared to media-only controls. Veratridine alonereduced viability of MDL-MB-231 cells by 15% (FIG. 1) compared to mediaalone-treated cells. In this initial experiment, ouabain combined withveratridine produced a 30% reduction in cell viability. In MCF-7 cells,none of the treatments affected cell viability (Data not shown). Thisexperiment was repeated on a 35 mm petri dish for video recording asabove.

EXAMPLE 5 In Vivo Targeted Osmotic Lysis in Breast Cancer Xenografts

As an in vivo model of cancer, we injected 4 million MDA-MB-231 cells,suspended in Matrigel, subcutaneously into the backs of 5 nude (J-NU)mice. Each mouse developed 0.75-1.2 cm tumors in 3-5 weeks. The micewere injected subcutaneously with 10 mg/kg ouabain or saline and thenafter 30 min anesthetized with 4% isoflurane. The tumors of the micewere exposed through a small incision in the skin. An anode and acathode were inserted into each tumor, and a train of 120 1 V DC pulses(10 ms, 2 pulses per second for 1 min) using the Grass SD9 stimulatorwas delivered through a copper anode and cathode. Some controls receivedno stimulation. A total of 11 tumors were tested. Three tumors were frommice treated with 10 mg/kg s.c. ouabain and electrically stimulated(O-ES experimental). Of the rest, two tumors were from a mouse treatedwith saline and not electrically stimulated (S-NS control), three werefrom mice treated with 10 mg/kg s.c. ouabain but not electricallystimulated (O-NS control), and three were from mice treated with salineand electrically stimulated (S-ES control). The electrical stimulationwas repeated 15 and 30 min later. One day later, mice were sacrificedwith an overdose of Na⁺-pentobarbital, perfused with 4% bufferedparaformaldehyde, and the tumors removed. The tumors were sectioned at 5μm and stained with hematoxylin and eosin. Of the three tumors in theexperimental group (the O-ES group), the tumor from one mouse showed 80%cell death after treatment. However, normal muscle taken from the baseof the tumor in the same mouse showed no sign of cell death (Photos notshown). The other two mice in the O-ES group had tumors that were nothighly vascularized, and thus the ouabain could not efficientlydistribute to the tumor cells. Consequently, no lysis was seen in thetumors of these two mice. None of the tumors in any of the three controltreatments showed any sign of cell lysis.

This experiment was repeated using 10 mice (2 per control group and 4 inthe experimental group.) Four million MDA-MB-231 cells suspended in 50%Matrigel were injected s.c. in J/Nu mice, and tumors allowed to growuntil visibly vascularized (reddish in appearance.) Stimulationparameters were 10 V DC, 1 ms pulses @ 200 pulses per second. Drugtreatment was again 10 mg/kg s.c. ouabain. One day later, animals weresacrificed, perfused, tumors removed, and prepared for hematoxylin andeosin staining Stained sections from each of the tumors were evaluatedby a trained histologist. As in the pilot experiment, none of thecontrols showed any sign of lysis. One control tumor showed centralnecrosis, which is common for fast-growing tumors. All four of thetumors treated with ouabain and electrical stimulation had an area ofcell lysis that was between 50% to 80% of the total tumor volume.

To demonstrate that there is no effect of the TOL treatment on healthy,non-cancerous cells, J/Nu mice were injected with 10 mg/kg ouabain, s.c.After 30 min, muscle, peripheral nerves, heart, and brain wereelectrically stimulated with the parameters in the previous experiments.These tissues were selected because they have the highest normalexpression of VGSCs, and would thus be expected to be most susceptibleto lysis of healthy cells. One day later, the animals were sacrificedwith an overdose of pentobarbital, and processed for hematoxylin andeosin staining None of these tissues showed signs of lysis.

EXAMPLE 6 Tumor Growth Following a Single TOL Treatment

To demonstrate an effect on post-treatment survival, 20 J/Nu mice weretreated with MDA-MB-231 cells as in the previous experiment. Whenvascularized tumors became apparent, mice were divided into 4 treatmentgroups: Saline-No stimulation; Saline-Stimulation (2×1 min electricalstimulation); Ouabain (10 mg/kg)-No stimulation; and Ouabain (10mg/kg)-Stimulation (2×1 min electrical stimulation). Only a singletreatment was given for each treatment group on Day 0 of FIG. 2. Tumorcross-sectional area was then measured with calipers every-other day forthree weeks. The tumor growth was expressed as a percent pre-treatmentsize and plotted against time. FIG. 2 shows the results of all fourgroups. As shown in FIG. 2, tumors on mice treated with ouabain andelectrical stimulation were 70% smaller (p<0.01) than tumors in thethree other groups.

In summary, we have demonstrated efficacy in both in vitro and in vivomodels of invasive carcinoma. We have shown the usefulness of bothelectrical and pharmacological stimulation of the cancer cells to induceTOL. We have demonstrated TOL in seven cell lines derived from fourdifferent tissue types. As little as 100-fold increase in sodiumchannels expression compared to normal tissue is sufficient to confersusceptibility to TOL treatment, although time-to-lysis is inverselyrelated to extent of sodium channel expression. We have demonstrated invivo that TOL does not affect normal tissue, even in those tissues thatnormally express relatively high concentrations of sodium channels.Finally, we have demonstrated that TOL can be induced using any drug orprocess that blocks sodium pumps.

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The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference arethe complete disclosures of the parent application, Ser. No. 13/552,909;and of the priority application, Ser. No. 61/510,258.

What is claimed:
 1. A method for treating cancer in a mammal; wherein,as compared to normal cells, cells of the cancer over-expressvoltage-gated sodium channels; said method comprising co-administeringto the cancerous cells a first agent and a second agent; wherein: (a)the first agent comprises a drug compound that decreases the activity orexpression of sodium, potassium-adenosine triphosphatase; (b) the secondagent stimulates the activity of voltage-gated sodium channels; (c) thecombined effects of the first agent on sodium, potassium-adenosinetriphosphatase and of the second agent on voltage-gated sodium channelsinduce, within one day of said co-administration, the osmotic lysis ofcancerous cells that over-express voltage-gated sodium channels; and (d)non-cancerous muscle cells, non-cancerous peripheral nerve cells,non-cancerous heart cells, and non-cancerous brain cells are notosmotically lysed following said co-administration.
 2. The method ofclaim 1, wherein the mammal is a human.
 3. The method of claim 1,wherein the cancer is selected from the group consisting of breastcancer, prostate cancer, small cell lung cancer, non-small cell lungcarcinoma, lymphoma, mesothelioma, neuroblastoma, glioma, neuroma,hepatic cancer, ovarian cancer, bladder cancer, pancreatic cancer,thyroid cancer, splenic cancer, stomach cancer, skin cancer, testicularcancer, renal cancer, oral cancer, and cervical cancer.
 4. The method ofclaim 1, wherein the cancer is breast cancer.
 5. The method of claim 1,wherein the cancer is prostate cancer.
 6. The method of claim 1, whereinthe cancer is colon cancer.
 7. The method of claim 1, wherein the canceris small cell lung cancer.
 8. The method of claim 1, wherein the canceris non-small cell lung cancer.
 9. The method of claim 1, wherein thefirst agent comprises one or more drug compounds selected from the groupconsisting of digoxin, digitoxin, digitalis, ouabain, oleandrin,dihydroouabain, ouabain octahydrate, ouabagenin, acetyldigitoxin,acetyldigoxin, lanatoside C, deslanoside, metildigoxin, gitoformate,oleandrigenin, bufotoxin, bufotalin, marinobufagenin, palytoxin;oligomycins, rutamycin, rutamycin B, strophanthin, k-β-strophanthin,strophanthidin, k-strophanthoside, cymarin, erysimoside (cardenolide),helveticoside, peruvoside, hypothalamic sodium, potassium-adenosinetriphosphatase inhibitory factor (HIF), the aglycone of HIF,arenobufagin, cinobufagin, marinobufagin, proscillaridin, scilliroside,and daigremontianin.
 10. The method of claim 1, wherein the first agentcomprises ouabain.
 11. The method of claim 1, wherein the first agentcomprises digitoxin.
 12. The method of claim 1, wherein the second agentcomprises a drug that stimulates the activity of voltage-gated sodiumchannels.
 13. The method of claim 1, wherein the second agent comprisesone or more drug compounds selected from the group consisting ofveratridine, veracevine, antillatoxin (ATX), ATX II, batrachotoxin,aconitine, grayanotox, Grayanotoxin III Hemi(ethyl acetate),Antillatoxinn, Nigericin, gramicidin, α-Pompilidotoxin,β-Pompilidotoxin, Hoiamide A, brevetoxin (PbTx-2), ciguatoxins, scorpionneurotoxin, Cypermethrin, Alphamethrin, and palytoxin.
 14. The method ofclaim 1, wherein the second agent drug comprises veratridine.
 15. Themethod of claim 1, wherein the second agent comprises an electricalcurrent.
 16. The method of claim 1, wherein the second agent comprisesultrasound.
 17. The method of claim 1, wherein the second agentcomprises a magnetic field.